In the past year, we continued to study Plasmodium falciparum genome diversity and its relationship with drug resistance, genetic recombination, gene expression regulation, and virulence using genetic mapping, microarray, gene knockout, genetic crosses, population genetics, and other approaches. The ultimate goals are to relate genetic polymorphisms to parasite biology and to develop novel approaches to control malaria. Using high-throughput chemical screening and genome-wide association analysis, we identified 32 highly active compounds against P. falciparum and many genetic loci and genes associated with differential chemical phenotypes (DCPs), defined as &#8805;5-fold differences in half-maximum inhibitor concentration (IC50) between parasite lines. We searched for compounds with common chemical response signatures in 61 parasite isolates and identified 44 compound clusters. We showed that several of these clusters act on a common target or response pathway. We also identified large numbers of drugs that were negatively correlated in responses among the parasites, suggesting candidates for effective drug combinations. For example, 43 drugs were significantly negatively correlated in response to chloroquine. We showed antimalarial drugs against pfmdr1 and pfcrt played a significant role in shaping current parasite populations and demonstrated that known drug resistance genes could be identified using genome-wide association analysis despite the presence of population structure. Known drug resistance genes (pfcrt, pfmdr1, and pfdhfr) were successfully associated with their respective drugs. We mapped 49 DCP compounds to 57 chromosomal loci/genes with LOD score&#8805;3 using 67 progeny from two genetic crosses and tested parasites with genetically modified pfcrt and pfmdr1 to conform that specific mutations in the genes indeed played a role in the differential sensitivities of many drugs. We then identified drugs targeting wild type or mutant pfcrt or pfmdr1 alleles and tested some drug combinations to show greatly reduced IC50 values to many drugs in the presence of a low dose chloroquine. These drug combinations could be targeting the Achilles heel of the parasite because both genes are essential to the parasite. This work was recently published in Science as a research article. The rodent malaria parasite Plasmodium yoelii is an excellent model for studying malaria disease phenotype because the genetic background of the host can be controlled using inbred mice;however, no genetic map is available today, partly due to the lack of an in vitro culture method for growing the parasite and the requirement for injecting a single parasite into a mouse to clone a progeny from a genetic cross. The absence of a genetic map impedes efforts to map genes and to assemble the parasite genome sequences. We performed 14 individual crosses involving three pairs of parental parasite clones/subspecies and developed a genetic map for Plasmodium yoelii to study disease phenotypes. We estimated recombination rate of the parasite for the first time and compared recombination frequency in different genetic backgrounds (crosses). We phenotyped (growth rate measurement) 25 progeny from one of the crosses and identified three loci linked to growth phenotype, including one on a locus on chromosome 13 and another on chromosome 10 that were linked to day 5 parasitemia and virulence. The locus on chromosome 10 could be the unknown factor implicated in previous studies and may partly explain the switches of parasite invasion preference. We identified a candidate gene encoding the P. yoelii nigeriensis erythrocyte binding-like protein (PyEBL) in the chromosome 13 locus. Our data suggest that a C741Y substitution plays a role in the unique growth phenotype of this subspecies. This work has recently published in PNAS. In our effort to study the mechanism of genetic recombination in this parasite, we used a tiling array and progeny from a P. falciparum genetic cross to investigate the parasites genetic recombination rate, recombination hotspot, and motifs associated with the hotspots. We found that the recombination rate in the parasite is higher than previously estimated and identified many recombination hotspots. From the hotspot sequences, we discovered several motifs that were enriched in the hotspots. This study represents the first investigation of recombination hotspots and motifs potentially associated with high recombination rate in malaria parasites. The hotspots and motifs provide important information for studying mechanism of genetic recombination in malaria parasites and possibly other microbes. This study was published in Genome Biology that turns out to be one of highly viewed articles. Several studies have shown differences in gene expression between pairs of isogenic parasites, but no changes in DNA sequences could be identified. We are still working in relationships between parasite nucleosome position and gene expression in various parasite developmental stages. In collaboration with Dr. Keji Zhao of NHLBI, we obtained nucleosome positions and mRNA sequences from different developmental stages of P. falciparum. Mistakes in predicted gene models and large numbers of antisense transcripts have been discovered. We are in the process of analyzing the data for publication.